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Background  Hearing loss is the most common sensory deficit in humans, affecting approximately 10% of the global population. In developed countries, one in every 500 individuals suffers from severe to profound bilateral sensorineural hearing loss. For those up to 5 years old, the proportion is higher, at 2.7 in 1000 individuals, and for adolescents the average is 3.5 in 1000. Among the causes of hearing loss, more than 50% are related to genetic factors. To date, nearly 150 loci and 64 genes have been associated with hearing loss. Mutations in the  GJB2  gene, which encodes connexin 26, constitute the main genetic cause. So far, more than 300 variations have been described in this gene.  As a response to the clinical and genetic heterogeneity of hearing loss and the importance of correct molecular diagnosis of individuals with hereditary hearing loss, this study worked in the optimization for a diagnostic protocol employing a high-throughput genotyping technology.    Methods  For this work, was used the TaqMan® OpenArray™ Genotyping platform. This is a high performance, high-throughput technology based on real-time PCR, which enables the evaluation of up to 3072 SNPs (Single Nucleotide Polymorphisms), point mutations, small deletions, and insertions, using a single genotyping plate. For the study, were selected the layout allowing to analyze 32 alterations in 96 individuals simultaneously. In the end, the generated results were validated by conventional techniques, as direct sequencing, Multiplex PCR and  RFLP-PCR .    Results  A total of 376 individuals were analyzed, of which 94 were healthy controls, totaling 4 plates in duplicate. All 31 of the changes analyzed were present in the nuclear genes  GJB2, GJB6, CRYL1, TMC1, SLC26A4, miR-96,  and  OTOF , and in the mitochondrial genes  MT-RNR1  and  MT-TS1 . The reactions were subsequently validated by established techniques (direct sequencing, multiplex PCR, and  RFLP-PCR ) that had previously been used to perform molecular screening of hearing loss at the Human Genetics Laboratory of the Center for Molecular Biology and Genetic Engineering (CBMEG), at the State University of Campinas (UNICAMP). In total, 11,656 genotyping reactions were performed. Of these, only 351 reactions failed, representing approximately 3.01% of the total. The average accuracy of genotyping using the OpenArray™ plates was 96.99%.    Conclusions  The results demonstrated the accuracy, low cost, and good reproducibility of the technique, indicating that the TaqMan® OpenArray™ Genotyping Platform is a useful and reliable tool for application in molecular diagnostic testing of hearing loss.

Optimization of simultaneous screening of the main mutations involved in non-syndromic deafness using the TaqMan® OpenArray™ Genotyping Platform